THE EFFECTS OF PHYSICAL AND CHEMICAL STRESS ON MEMBRANE PERMEABILITY
The aims of this experiment were to:
• Investigate temperature and solvents on beetroot membrane integrity
• To use skills in spectrophotometry to measure pigment leakage
Cells membranes are crucial for life, they define cell boundary. They are also known as plasma membranes or plasmalemma. The function of the cell membrane is to support the cell and regulate what enters and exits the cell. It also enables cell signalling and cell recognition and furthermore, determines organelle boundaries inside the cell.
The cell membrane is composed of;
1. Phospholipids which are arranged as a phospholipid bilayer, this is created due to the phospholipids containing a hydrophilic head and a hydrophobic tail. The tails bundle together and only present the polar heads to the water. (Unknown, 2003)
2. Proteins which are inserted in the bilayer. There are two types of proteins such as integral proteins for example connexions. The other type being peripheral proteins which may include clatherin adaptor protein which can separate from the membrane.
3. Cholesterol and other sterols make the membrane less permeable to hydrophilic molecules.
• The presence of hydrogen bonding of water molecules holds the 2 layers together.
• The fluid structure means the proteins and lipids in the membrane can move with ease.
According to (Chandler, 2017), When cell membranes are exposed to extremely high temperatures the cell membrane becomes more fluid, this occurs when the fatty acid tails of the phospholipids become less rigid and enables more movement of proteins and other molecules in and through the membrane. This can change the permeability of the cell, potentially enabling possible harmful materials into the cell. Both integral and peripheral proteins in the membrane can also become damaged, extremely high temperatures could cause them to break down or even denature.
On the other hand, according to (Chandler,2017), a decrease in temperature can have a negative effect on cell membranes and cells. At really low temperatures, the fatty acid tails of the phospholipids move less and become more rigid (less permeable). This causes a decrease in the overall fluidity of the cell membrane. This could lead to potentially blocking essential materials from entering such as oxygen or glucose. In extremely low temperatures, liquids within the cell begin to freeze, forming crystals that pierce the membrane and therefore could potentially kill the cell.
The fluid mosaic model is a mosaic of proteins which floats in or on the fluid lipid layer just like icebergs in the sea.
The deep red pigment of beetroot is due to the presence of a class of compounds called betacyanins. This pigment is found deep within membrane-bound vacuoles which are surrounded by the tonoplast membrane inside the cell. If the membrane is somehow amended leakage (diffusion) of betacyanin is induced.
Beetroot has a presence of 2 Betacyanins, Betanin and a derivative. (Goncalves et al, 2012)
When the cell membrane undergoes chemical stress this causes interruption to the homeostasis balance inside the cell which causes an increase in diffusion.
o Fresh beetroot
o Frozen beetroot
o Spectrophotometer (450nm)
o White tile
o Razor blade
o Cork borer (10mm diameter)
o 7 test tubes
o Deionised water
o water baths
o 25cm3 measuring cylinder
o Acetone (1%, 25%, 50%)
o Methanol (1%, 25%, 50%)
To begin with all materials mentioned above were collected. The fresh beetroot was thoroughly washed. 13 cylinders was then removed from the fresh beetroot using a cork borer of approximately 10mm diameter, another cylinder was bored out of the frozen beetroot and all cylinder were cut to the same length at approximately 1.5cm to 2cm each, before continuing it was essential that all the skin was removed and a clean cut was left at each end. All cylinders were washed under a running tap for 5 minutes in order to remove the pigment from the surface.
The first part of the experiment was the effect of heat, using forceps 1 cylinder was gently held in a water bath at 70oc for 60 seconds it was then removed and placed in a test tube which contained 15ml distilled water and was allowed to stand at room temperature for 45 minutes. This procedure was repeated under 5 other temperatures, (65oc, 60oc, 55oc and 50oc. As a control, an unheated cylinder was placed into a test tube filled with 15ml of distilled water at room temperature. After 45 minutes, all of the beetroot cylinders were removed using forceps and the pigment leakage was measured using a spectrophotometer set at 460nm and then recorded into a table.
The second part of the experiment was the effect of freezing. The cylinder that was previously extracted from the frozen beetroot was briefly washed under a running tap and then immediately placed into a test tube containing 15ml of distilled water and was left at room temperature for 45 minutes. Once the 45 minutes was completed the beetroot cylinder was carefully removed using forceps and the pigment leakage was measured in a spectrophotometer at 460nm and recorded into to a table. The third part of the experiment was the effect of organic solvent, 10ml of (1% acetone, 25% acetone, 50% acetone, 1% methanol, 25% methanol, 50% methanol) were supplied in bottles. 1 washed beetroot cylinder was placed into each cylinder bottle and were left for 20 minutes. Once the 20 minutes was over each bottle was quantified regarding the membrane permeability according to the betacyanin. Each bottle was given a value, 1 being the darkest and 6 being the lightest.
TABLE 1: EFFECT OF HEATING AND FREEZING
ABSORBANCE AT 460nm
The table above displays the effects of difference temperatures on the pigment leakage of the beetroot cells.
GRAPH 1: THE EFFECT OF HEATING AND FEEZING ON MEMBRANE INTEGRITY
The graph above shows that at 70oc the absorbance peaks to 0.904nm and then at 650c the absorbance drops to 0.189nm. At 60oc the absorbance goes back up to 0.224. From 55oc a trend starts to occur, that as the temperature decreases the absorbance also decreases, however at -20oc the pigment leakage peaks up to 1.310nm.
TABLE 2: EFFECT OF CHEMICAL SOLVENTS ON MEMBRANE INTEGRITY
DARKEST – LIGHTEST
This table demonstrates that the 1% solvents have the greatest pigment leakage, whereas the 50% solvents have the least pigment leakage.
After viewing the data from table 1 it could be said that as the temperature of the water bath decreased the pigment leakage decreased as it went from 0.904nm at 70oc to 0.050nm at 20oc. When there is a low temperature surrounding the cell membrane such as 20oc the cell membrane becomes more permeable as the absorbance measured for this value was 0.065nm. These results were not as expected because according to (Chandler, 2017) the fatty acid tails of the phospholipids move less and become more rigid. Meaning the cell membrane becomes less permeable. Furthermore, when extreme cold temperatures were exposed to the cell membrane such as -20oc the absorbance value peaked right up to 1.310nm, this could be due to the fact that In extremely low temperatures, liquids within the cell begin to freeze, forming crystals that pierce the membrane and therefore could potentially kill the cell.
When there is an increase in temperature such as 70oc around the cell the cell membrane becomes slightly more permeable therefore allowing more scope as to what can enter and exit the cell through the membrane
After processing the data from the second table it can be said that the 50% solutions produced a greater pigment leakage due to more betacyanin being diffused out of the beetroot meaning this was the darkest solution. This could be due to the fact that cell membranes are very receptive and the lipids in the bilayer liquefy because the solvent is so strong and the cell becomes more permeable due to the loss of lipids in the phospholipid bilayer. It was also clear that the 1% solutions produced the smallest pigment leakage due to hardly any betacyanin being leaked out of the beetroot. Low solvent concentrations do not dissolve as many lipids therefore the cell membrane becomes slightly permeable but does not allow as many materials in and out of the cell as the high percentage solvent concentrations.
Potential errors that may have occurred in this procedure was that the beetroot cylinders may not have been left in the distilled water for exactly 45 minutes.
Another error may be that the beetroot cylinders were not fully submerged in the water baths for the correct period of time.
Another error could be that the cuvette was placed into the spectrophotometer incorrectly, meaning the arrow of the cuvette was not facing the light.
Chandler, S. (2017). The Effect of Temperature on Cell Membrane Permeability. Available: https://sciencing.com/effect-temperature-cell-membranes-5516866.html. Last accessed 20th Nov 2017.
Goncalves, L et al (2012). Food Chemistry. Unknown: Elsevier. 231-238.
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